Ac-SDKP Attenuates Epithelial-mesenchymal Transition Regulated By Hedgehog Signaling In Alveolar Type Ⅱ Epithelial Cells

Objectives To explore the effect and mechanism of N-acetyl-seryl-aspartyl-lysyl-proline（Ac-SDKP）on silica induced epithelial-mesenchymal transition（EMT）in alveolar epithelial type Ⅱ cells regulated by Hedgehog（HH）signaling pathway.Methods The rats were divided into three groups: 1)control group;2)silicosis model group;3)Ac-SDKP post-treatment group.The MLE-12 cells were cultured and divied as 1)control group,SiO₂ group,Ac-SDKP group and SiO₂ +Ac-SDKP group;2)solvent control group,SiO₂ induction group,GDC-0449 group and SiO₂ +GDC-0449 group;3)solvent control group,SiO₂ induction group,GANT61 group and SiO₂ + GANT61 group.The collagen deposition was detected by Sirius red staining.The localization and expression of α-smooth muscle actin（α-smooth muscel actin,α-SMA）and sonic hedgehog factor（sonic hedgehog,SHH）were detected by immunohistochemical（IHC）staining.The expression of E-cadherin,the co-expression of vimentin and ATP binding box transporter A3（ABCA3）,SHH and E-cadherin,SHH and vimentin was detected by immunofluorescence（IF）staining.The expression of Collagen type I（Col Ⅰ）,α-SMA,E-cadherin,SHH,Smoothened（SMO）,（Glioma-associated oncogene homolog,Gli）1 and Gli2 were measured by western blot.Results 1 Compared with the control group,the expression of E-cadherin was decreased in SiO₂ group by IF staining,accompanied with increasing expression of α-SMA measured by IHC staining.Western blot results showed that the level of E-cadherin was downregulated,and the levels of Col Ⅰ and α-SMA were up-regulated in SiO₂ group compared to control group（P<0.05）.2 Compared with the control group,the expression of SHH,SMO,Gli1,and Gli2 were all increased in MLE-12 cells exposed to 50 μg/m L SiO₂（P<0.05）.3 Pretreatment with GDC-0449 or GAN61 resulted in increasing of E-cadherin,and decreasing of Col I and α-SMA.4 Compared with SiO₂ group,the expression of Ecadherin was increased in SiO₂+Ac-SDKP group measured by IF staining,accompanied with decreasing expression of α-SMA observed by IHC staining.The level of E-cadherin was increased in SiO₂+Ac-SDKP group compared to silica group,with a decreasing level of Col I,α-SMA,SHH,SMO,Gli1,and Gli2 which measured by western blot（P<0.05）.5 The sirus red staining showed that silicotic lesions could be observed in rats exposed to silica,and the number and area of the silicotic lesions were decreased in Ac-SDKP posttreatment group.Compared with control group,the level of Col Ⅰ and α-SMA were increased in silicotc rats with a down-regualtion of E-cadherin.Post-treatment with AcSDKP could attenuate the silica-induced changes in rats（P<0.05）.The expression of SHH was observed in silicotic lesions by IHC staining.IF staining showed that co-expression of SHH with E-cadherin,and SHH with vimenitn were found in lungs of rats.The level of SHH,SMO,Gli1,and Gli2 were reduced in Ac-SDKP post-treatment group compared with silicotic group（P<0.05）.Conclusions Ac-SDKP inhibited EMT regulated by HH signaling in alveolar epithelial type Ⅱ cells to attenuate silicosis.Figure 16;Table 8;Reference 156...